Escherichia coli producing capsanthin and application thereof

Abstract

The invention discloses an escherichia coli for producing capsanthin, which is classified and named as Escherichia coli Cap-OUC-CHB, is preserved in the China Center for Type Culture Collection (CCTCC), has the preservation number of CCTCC NO: M 20241969, and is preserved on September 13, 2024. The invention also discloses an application of the strain in preparation of capsanthin. The invention also discloses a construction method of the recombinant engineering bacterium for producing capsanthin. According to the escherichia coli capable of producing capsanthin, high yield of capsanthin and capsanthin can be achieved, the accumulation amount of capsanthin and capsanthin can reach 6.77 mg / g DCW and 2.18 mg / g DCW, and the escherichia coli has the advantages of being fast in growth and reproduction, high in product concentration, stable and reliable in reaction and the like, has the potential of industrial production and has good application prospects.

Description

Technical Field

[0001] The invention relates to a capsanthin-producing Escherichia coli and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Capsanthin is a type of tetraterpenoid natural carotenoid extracted from ripe red peppers. It is listed as a Class A colorant by the Food and Agriculture Organization of the United Nations and the World Health Organization, which means that it can be added in unlimited quantities during cooking. Capsanthin is mainly composed of two pigments: capsanthin and capsorubin, of which capsanthin is the main component of capsanthin. At present, capsanthin has been verified to have important physiological activities, such as reducing fat absorption, increasing fat consumption, reducing insulin resistance, anti-colon cancer, and inhibiting the growth of lung cancer and triple-negative breast cancer cells. In short, capsanthin is a natural compound with great application prospects in the fields of food and medicine.

[0003] However, the natural content of capsanthin is very low, only 1.32‰~1.34‰, accounting for 56.06%~57.14% of the total carotenoids in peppers, of which the average content of capsanthin is 1.27‰ and that of capsanthin is only 0.586‰~0.672‰. In addition to the low natural content, the bioavailability of its esterified form is low, which seriously limits its application in the medical field. Moreover, the pepper meal extracted with chemical organic reagents is mainly cellulose and hemicellulose, which is difficult to reuse as poultry and livestock feed because of its poor palatability. In addition, due to the light and heat sensitivity of capsanthin, the equipment and temperature requirements for extraction with organic reagents are high.

[0004] With the development of science and technology such as synthetic biology and genetic engineering, microbial fermentation has gradually become an effective way to produce natural products. However, the difficulty in constructing recombinant engineered bacteria that produce capsanthin is that the synthesis pathway of capsanthin is complex, and it takes multiple transformations to recombinate the expression genes of the enzymes involved in the synthesis pathway into the engineered bacteria, which is difficult and has a low success rate; and after the heterologous genes are transferred to enable the genetically engineered bacteria to synthesize the product, the intermediate strains need to be transformed and screened multiple times to obtain high-yield genetically engineered bacteria. At present, there are no reports on the construction of recombinant engineered bacteria that produce capsanthin. Summary of the invention

[0005] In view of the above prior art, the present invention constructs a recombinant engineering bacterium through genetic engineering, and screens and obtains a high-yield capsanthin Escherichia coli. The present invention also provides the use of the capsanthin-producing Escherichia coli in the preparation of capsanthin.

[0006] The present invention is achieved through the following technical solutions: A strain of Escherichia coli producing capsanthin, classified and named Escherichia coli Cap-OUC-CHB, is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241969 and a deposit date of September 13, 2024.

[0007] The biological characteristics of the Escherichia coli producing capsanthin are as follows: facultative anaerobic Escherichia coli, cultured on LB solid medium for 12 hours at 37°C, the colonies are white, smooth in surface and neat in edge. Aerobic fermentation is used in liquid fermentation.

[0008] The application of the capsanthin-producing Escherichia coli in the preparation of capsanthin.

[0009] Furthermore, the capsanthin is selected from any one or two of capsanthin and capsorubin.

[0010] Furthermore, in a specific application, Escherichia coli producing capsanthin is cultured and capsanthin is extracted.

[0011] Furthermore, the specific method of culturing Escherichia coli producing capsanthin can be: inoculating the seed liquid of Escherichia coli producing capsanthin into the self-induction fermentation medium, with an inoculation amount of 2% (volume percentage), and inducing fermentation in a shaking table at 37°C and 220rpm for 48 to 96 hours (preferably 72 hours).

[0012] Furthermore, the self-induction fermentation medium is selected from ZYP-5052 medium (a commercial medium available in the prior art).

[0013] Furthermore, the self-induction fermentation medium contains 20 mM sodium formate; at the 36th hour of fermentation, 20 mM arabinose is added to the fermentation broth for induction.

[0014] Furthermore, the specific method of extracting capsanthin can be: taking the cultured fermentation broth, centrifuging and discarding the supernatant, adding an appropriate amount of acetone to the precipitate, extracting, centrifuging, filtering and removing impurities from the supernatant to obtain an extract, which contains capsanthin and capsanthin.

[0015] Furthermore, the amount of acetone added is: 1 mL of acetone per 10 mL of fermentation liquid.

[0016] The present invention also provides a method for constructing a recombinant engineering bacterium producing capsanthin, the steps of which are as follows: (1) Constructing a recombinant Escherichia coli producing zeaxanthin as a chassis cell: using E. coli BL21 (DE3) (a host strain suitable for expressing foreign proteins in the prior art) as a starting strain, using gene editing vectors pHCY-25A and pHCY-26D to knock out the trxb gene, and then using pTrc99a as an expression vector to transfer the CrtE, CrtB, CrtI, CrtY and CrtZ genes, constructing and screening recombinant engineered bacteria (after each transformation, the transformant with the highest yield is selected as the host for the next transformation, the same below); the nucleotide sequence of N20 targeting trxb used to knock out the trxb gene is shown in SEQ ID NO.1; the nucleotide sequences of the CrtE, CrtB, CrtI, CrtY and CrtZ genes are shown in SEQ ID NOs.2 to 6, respectively; (2) Constructing a recombinant Escherichia coli that produces violaxanthin as a chassis cell: using pRSFDuet-1 as an expression vector, the Zep gene was transferred into the recombinant engineered bacteria constructed in the above step (1); then, using pTrc99a-EBIYZ as an expression vector, the Trx-m1, Trx-m2 and Trx-m4 genes were transferred to construct and screen the recombinant engineered bacteria; the nucleotide sequences of the Zep, Trx-m1, Trx-m2 and Trx-m4 genes are shown in SEQ ID NOs. 8, 10, 11 and 12, respectively; (3) Construction of recombinant Escherichia coli producing capsanthin and capsanthin: Using pRSFDuet-ZEP as an expression vector, the ccs gene with the scaffold protein SpyCatcher connected to the 5' end and the β-cav1 gene with SpyTag connected to the 5' end were transferred into the recombinant engineered bacteria constructed in the above step (2); then, using pACYCDuet-1 as an expression vector, the Cpn60α, Cpn60β and Cpn20 genes were further transferred; then, using pCDFDuet-1 as an expression vector, the FDHm and Fre genes were further transferred to construct and screen recombinant Escherichia coli producing capsanthin and capsanthin; the nucleotide sequences of the SpyCatcher-ccs, SpyTag-β-cav1, Cpn60α, Cpn60β, Cpn20, FDHm and Fre genes are shown in SEQ ID NOs. 13 to 19, respectively.

[0017] The recombinant engineered bacteria are constructed using the above-mentioned method for constructing recombinant engineered bacteria that produce capsanthin.

[0018] The construction method of the present invention has the following beneficial effects: (1) Zeaxanthin epoxidase ZEP from the plastids of Haematococcus lacustrine was introduced into the zeaxanthin-producing chassis cells. By knocking out the trxb gene on the Escherichia coli genome, an oxidative catalytic environment was created for ZEP. The highly active form of ZEP can promote the conversion of zeaxanthin into antheraxanthin and violaxanthin, providing sufficient precursors for the next step of synthesizing capsanthin and capsorubin.

[0019] (2) Capsanthin synthase CCS is derived from pepper chloroplasts. The present invention uses Spy scaffold protein to assemble homotrimers of CCS, which mimic its structure in a quasi-physiological state. At the same time, molecular chaperones Cpn60α, Cpn60β and Cpn20 derived from pepper chloroplasts are introduced to promote the folding of CCS. FADH2 is an essential cofactor for CCS, and FDHm can catalyze sodium formate.

[0020] The capsanthin-producing Escherichia coli of the present invention can achieve high production of capsanthin and capsuorubin (the accumulation amounts of capsanthin and capsuorubin can reach 6.77 mg / g DCW and 2.18 mg / g DCW), has the advantages of fast growth and reproduction, high product concentration, stable and reliable reaction, etc., has the potential for industrial production, and has good application prospects.

[0021] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The capsanthin-producing Escherichia coli of the present invention is classified and named Escherichia coli Cap-OUC-CHB, and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20241969, a preservation date of September 13, 2024, and a preservation address of: China Center for Type Culture Collection of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0023] Figure 1 :Schematic diagram of the synthesis pathway of capsanthin and capsanthin.

[0024] Figure 2 : Schematic diagram of zeaxanthin production by recombinant Escherichia coli EcZea.

[0025] Figure 3 :Schematic diagram of the effect of zeaxanthin epoxidase from different sources on violaxanthin production.

[0026] Figure 4 :Schematic diagram of the effect of knocking out the trxb gene on violaxanthin production.

[0027] Figure 5 : Schematic diagram of the effect of co-expression of thioredoxin on violaxanthin production.

[0028] Figure 6 :Schematic diagram of the effects of different assembly methods on capsanthin production.

[0029] Figure 7 : Schematic diagram of capsanthin production at different induction times.

[0030] Figure 8 : Schematic diagram of capsanthin production at different sodium formate concentrations. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0032] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0033] The specific steps of the Escherichia coli CaCl2 chemical transformation method adopted in the present invention are as follows: (1) Take the engineered bacteria from -70℃ and inoculate them into LB medium at 37 o C for 12-16 h; (2) Transfer the culture medium to a centrifuge tube, place on ice for 10 minutes, and then centrifuge at 4°C and 4000 rpm for 10 minutes. Discard the supernatant and gently suspend the cells with 10 ml of pre-cooled 0.05 mol / L CaCl2 solution. Place on ice for 15 to 30 minutes and then centrifuge at 4°C and 4000 rpm for 10 minutes. (3) Discard the supernatant, add 4 mL of pre-cooled 0.05 mol / L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to obtain the competent cell suspension, which is then stored at -70°C for later use; (4) Take 200 μL of competent cell suspension from the -70°C refrigerator, thaw on ice, add plasmid DNA solution (content not exceeding 50 ng, volume not exceeding 10 μL), shake gently, place on ice for 30 min, heat shock in a 42°C water bath for 90 s, and quickly cool on ice for 3 to 5 min. (5) Add 1 ml of LB liquid medium (without antibiotics) to the tube, mix well, and culture at 37°C with shaking for 1 hour to allow the bacteria to return to normal growth and express the antibiotic resistance gene encoded by the plasmid; (6) After shaking the above bacterial solution, spread 100 μL on a screening plate containing the corresponding antibiotic and incubate it inverted at 37°C for 12 to 16 h.

[0034] The shake flask fermentation method used in the present invention is as follows: 50 mL of ZYP-5052 culture medium is added to a 250 mL shake flask, the engineered bacteria seed solution is inoculated at a 2% inoculation rate, and cultured at 37°C and 220 rpm. Unless otherwise specified, the fermentation time is 72 h.

[0035] The specific steps of the gene editing method used in the present invention are: (1) The BL21 (DE3) strain was prepared into chemically competent cells, and the pHCY-25A plasmid was transformed into it to obtain the BL21 (DE3) / pHCY-25A strain; (2) Prepare the BL21 (DE3) / pHCY-25A strain into chemically competent cells, and add kanamycin (Kan) and glucose to the culture medium; (3) Transform the pHCY-26D plasmid into BL21 (DE3) / pHCY-25A competent cells, apply to LB plates (Amp+Kan+glucose), and culture at 30°C; (4) When the colonies grow up, pick 3 to 4 colonies from the plate and inoculate them into 2 mL LB medium, add 2 μL of Amp (100 mg / ml) and 2 μL of Kan (50 mg / ml), and culture at 30°C for 2 h. Then add 20 μL of IPTG (100 mM), and culture at 30°C for 1 h. Then add 40 μL of L-arabinose (1 M), and culture at 30°C for 3 h. (5) Dilute the bacterial solution 100 times and 1000 times, then take 100 μL of the diluted bacterial solution and apply it to LB plate (Amp+Kan+L-arabinose), incubate at 30°C, and perform PCR verification after the colonies grow; (6) The lost gene editing plasmid is used for further chassis cell construction. The correct colony is inoculated into 2 mL of LB medium without antibiotics and cultured at 37°C overnight. Then 2 μL of the bacterial solution is streaked on an LB plate containing 1% sucrose and cultured at 37°C. Most of the colonies grown on the plate have lost the pHCY-25A plasmid and the pHCY-25D plasmid. This tube of antibiotic-free bacterial solution can be used for other experiments.

[0036] The carotenoid extraction method adopted in the present invention is as follows: 10 mL of fermentation liquid is placed in a 50 mL centrifuge tube, centrifuged at 12000 rpm for 2 min, the supernatant is discarded, 1 mL of physiological saline is taken to resuspend the precipitate, and the precipitate is transferred to a 1.5 mL grinding tube, 1 mL of acetone and an appropriate amount of zirconium oxide grinding strain are added, and the supernatant is drawn by a syringe after grinding for 15 min, and impurities are removed by filtering with a 0.22 μm nylon filter membrane to obtain a total carotenoid extract.

[0037] The method for performing HPLC analysis on the extract of the present invention is as follows: C18 chromatographic column (250 mm×4.6 mm, 5 μm), column temperature 30°C, injection volume 10 μL, total flow rate 0.5 mL / min, gradient elution method, mobile phase A is isopropanol (chromatographic grade); mobile phase B is 80% acetonitrile (i.e., 200 mL of chromatographic grade water is added to 800 mL of chromatographic grade acetonitrile), and the concentration of pump B changes with time as follows: 0-25 min, 100%-37.5%; 25-45 min, 37.5%-37.5%; 45-47 min, 37.5%-100%; 47-57 min, 100%-100%; and carotenoids are detected at 472 nm.

[0038] In the present invention, the synthesis pathway of capsanthin is as follows Figure 1 shown.

[0039] The meanings of the English abbreviations involved in the present invention are as follows: CrtE: Geranylgeranyl pyrophosphate synthase.

[0040] CrtB: phytoene synthase.

[0041] CrtI: lycopene dehydrogenase.

[0042] CrtY: lycopene cyclase.

[0043] CrtZ: β-carotene hydroxylase.

[0044] trxB: thioredoxin reductase.

[0045] Trx-m1 / m2 / m4: thioredoxin m1 / m2 / m4.

[0046] ZEP: zeaxanthin hydroxylase.

[0047] CCS: capsanthin synthase.

[0048] FDHm: formate dehydrogenase.

[0049] Fre: flavin reductase.

[0050] DMAPP: dimethylallyl pyrophosphate.

[0051] FAD: flavin adenine dinucleotide.

[0052] FADH2: flavin adenine dinucleotide reduced.

[0053] SpyCatcher: A scaffold protein from Streptococcus pyogenes, responsible for the assembly of capsanthin synthase in this invention.

[0054] SpyTag: A scaffold protein from Streptococcus pyogenes, used in conjunction with SpyCatcher in the present invention.

[0055] β-cav1: A scaffold protein used in conjunction with SpyCatcher and SpyTag.

[0056] Cpn60α: a molecular chaperone from plant chloroplasts, subunit α.

[0057] Cpn60β: a molecular chaperone from plant chloroplasts, subunit β.

[0058] Cpn20: a molecular chaperone from plant chloroplasts.

[0059] Lyc: lycopene.

[0060] β-Car: β-carotene.

[0061] Zea: zeaxanthin.

[0062] Ant: anther xanthin.

[0063] Vio: Violaxanthin.

[0064] Cap: Capsanthin.

[0065] Cab: Capsanthin.

[0066] Amp: Ampicillin antibiotic.

[0067] Kana: kanamycin.

[0068] Smr: Spectinomycin.

[0069] Chl: chloramphenicol.

[0070] IPTG: isopropyl-β-D-thiogalactoside.

[0071] Example 1 Construction of recombinant engineered bacteria producing zeaxanthin First, the trxb gene on the E. coli genome was knocked out. The method used was CRISPR / Cas9, and five heterologous enzymes, CrtE, CrtB, CrtI, CrtY and CrtZ, involved in the biosynthesis pathway of zeaxanthin were transferred into this chassis cell. The expression genes of these five heterologous enzymes were sequentially connected to the pTrc99a vector to construct a chassis strain producing zeaxanthin. The specific process is described below.

[0072] 1. Construction of plasmid The gene knockout plasmid pHCY-26D-trxB was constructed, and the N20 sequence targeting trxB (as shown in SEQ ID NO.1) was constructed into the vector.

[0073] The CrtE, CrtB, CrtI, CrtY and CrtZ genes from Pantoea ananas were optimized according to the codon preference of Escherichia coli. The optimized nucleotide sequences are shown in SEQ ID NOs. 2 to 6, and Beijing Liuhe BGI Genomics Co., Ltd. was commissioned to artificially synthesize these gene fragments.

[0074] Using the synthesized gene fragment as a template, corresponding primers and P525 enzyme were used for PCR amplification to obtain a large number of target gene fragments.

[0075] Using plasmid pTrc99a as a template, primers and P525 enzyme were added for PCR amplification to obtain a plasmid backbone, which was recovered using a PCR recovery kit. The target gene fragment and the plasmid backbone were connected using homologous recombination seamless splicing technology to obtain plasmid pTrc99a-EBIYZ.

[0076] 2. Construction of zeaxanthin-producing engineered bacteria (1) Knockout of the trxb gene: pHCY-25A-trxB and pHCY-26D were sequentially transferred into BL21 (DE3) according to the above gene editing method, and L-arabinose was used to induce expression to knock out the trxb gene. After verification by PCR and sequencing, the gene editing plasmid was lost in the antibiotic-free LB medium.

[0077] (2) The pTrc99a-EBIYZ plasmid was transformed into the engineered bacterium BL21 (DE3)-Δtrxb using the CaCL2 chemical transformation method to obtain a recombinant engineered bacterium that produces zeaxanthin, which was named EcZea.

[0078] (3) The recombinant engineered bacteria EcZea were subjected to shake flask fermentation, extraction and HPLC detection. The dry weight yield of Zea-1 was: the carotenoid content per unit weight of the bacteria, in mg / g DCW (DCW, Dry Cell Weight), such as Figure 2As shown, the results showed that the strain could produce 0.3 mg / g DCW of lycopene, 1.5 mg / g DCW of β-carotene and 9.98 mg / g DCW of zeaxanthin. The recombinant engineered bacteria Zea almost only produced zeaxanthin and accumulated very little precursor substances, which could be further used for the construction of violaxanthin-producing chassis cells.

[0079] Example 2 Construction of recombinant engineering bacteria producing violaxanthin The conversion of zeaxanthin to violaxanthin requires the catalysis of zeaxanthin epoxidase. In this example, zeaxanthin epoxidases from different sources were introduced into the chassis cells of Example 1 to screen chassis cells with high violaxanthin production. Thioredoxins from different sources were also introduced to increase the stability of zeaxanthin epoxidase. The expression genes of these three heterologous enzymes were sequentially connected to the pTrc99a vector to construct a chassis strain producing violaxanthin. The specific process is described below.

[0080] 1. Construction of plasmid Zep3 from Phaeochromis tricornutum, Zep from Haematococcus pluvialis, TrxA from endogenous Escherichia coli, and Trx-m1, Trx-m2, and Trx-m4 from Arabidopsis thaliana were optimized according to the codon preference of Escherichia coli after removing the transit peptide sequence. The optimized nucleotide sequences are shown in SEQ ID NOs. 7 to 12, respectively. Beijing Liuhe BGI Genomics Co., Ltd. was commissioned to artificially synthesize these gene fragments. Among them, Zep3 and Zep were constructed on the pRSFDuet-1 vector, respectively, to obtain pRSFDuet-Zep3 and pRSFDuet-Zep plasmids, and TrxA, Trx-m1, Trx-m2 and Trx-m4 were constructed separately and in combination into the pTrc99a-EBIYZ vector to obtain pTrc99a-EBIYZ-TrxA, pTrc99a-EBIYZ-Trxm1, pTrc99a-EBIYZ-Trxm2, pTrc99a-EBIYZ-Trxm4 and pTrc99a-EBIYZ-Trxm1 / 2 / 4 plasmids.

[0081] Using the synthesized gene fragment as a template, corresponding primers and P525 enzyme were used for PCR amplification to obtain a large number of target gene fragments.

[0082] Using plasmids pRSFDuet-1 and pTrc99a-EBIYZ as templates, primers and P525 enzyme were added for PCR amplification to obtain the plasmid backbone, which was recovered using a PCR recovery kit. The target gene fragment and the plasmid backbone were connected using homologous recombination seamless splicing technology to obtain the corresponding plasmid.

[0083] 2. Construction of violaxanthin-producing engineered bacteria (1) The pRSFDuet-Zep3 and pRSFDuet-Zep plasmids were respectively transferred into BL21 (DE3) without knocking out the trxb gene by CaCL2 chemical transformation to obtain the violaxanthin-producing engineered bacteria EcVio-1 and EcVio-2. After screening the high-violaxanthin-producing strain, in order to explore the effect of cytoplasmic redox potential on ZEP activity, the corresponding plasmid pRSFDuet-Zep in the high-violaxanthin-producing strain EcVio-2 was transferred into the EcZea strain in Example (1) to obtain the strain EcVio-3. After further screening of high-yield strains, pTrc99a-EBIYZ-TrxA, pTrc99a-EBIYZ-Trxm1, pTrc99a-EBIYZ-Trxm2, pTrc99a-EBIYZ-Trxm4 and pTrc99a-EBIYZ-Trxm1 / 2 / 4 plasmids were introduced to replace the original pTrc99a-EBIYZ plasmid, and they were named EcVio-4, EcVio-5, EcVio-6, EcVio-7 and EcVio-8 respectively. Finally, the strain with the highest violaxanthin production was determined through comparison.

[0084] (2) The recombinant engineered bacteria in (1) were subjected to shake flask fermentation, extraction and HPLC detection, such as Figure 3 As shown, ZEP from Haematococcus pluvialis has higher catalytic activity, and strain EcVio-2 can produce 2.3 mg / g DCW of violaxanthin. Compared with the blank strain without knocking out the trxb gene, the knockout of the trxb gene in the EcZea strain in Example 1 blocked the transmission of reducing power on the cytoplasmic side, making the entire cytoplasmic side present a high redox potential state, promoting Zep oligomerization into a highly active form with stronger catalytic ability, such as Figure 4 As shown, strain EcVio-3 can produce 13.1 mg / g DCW of violaxanthin. Furthermore, based on strain EcVio-3, the effects of co-expression of thioredoxin from different sources on the accumulation of violaxanthin were compared, such as Figure 5 As shown in the figure, the strain EcVio-8 co-expressing Trxm1 / 2 / 4 from Arabidopsis thaliana obtained 16.8 mg / g DCW of violaxanthin and accumulated only 0.4 mg / g DCW of its precursor zeaxanthin. This strain not only produced high levels of violaxanthin but also had almost no precursor accumulation, laying the foundation for further construction of high-yield pepper red engineering bacteria.

[0085] Example 3 Construction of recombinant engineered bacteria producing capsanthin and improvement of yield Example 2 obtained a chassis cell with high production of violaxanthin, and based on the transformant EcVio-8, CCS was assembled into a multimer. In order to direct the product metabolic flux of ZEP to CCS, SpyCatcher-CCS and SpyTag-ZEP were constructed for assembly. In order to mimic the quasi-physiological state of CCS, SpyCatcher-CCS and SpyTag-β-cav1 were constructed to assemble CCS homotrimers and localize them to the cytoplasmic membrane. In addition, the molecular chaperones Cpn60α, Cpn60β and Cpn20 genes derived from Arabidopsis thaliana chloroplasts were introduced to promote the folding of CCS, and the FDHm and Fre genes were introduced to provide CCS with sufficient reduced cofactor FADH2 to further increase the yield. The specific process is shown below.

[0086] 1. Construction of plasmid The ccs gene is from pepper, and the transit peptide sequence is predicted and removed. SpyCatcher-ccs was connected to the second multiple cloning site after zep (using the pRSFDuet-Zep vector as a template), driven by the T7 lac promoter, and SpyTag was added in front of zep by homologous recombination, thus generating the plasmid pRSFDuet-SpyTag-ZEP-SpyCatcher-CCS. SpyCatcher-ccs and SpyTag-β-cav1 genes were sequentially connected to the back of the Zep gene (using the pRSFDuet-Zep vector as a template), among which SpyCatcher-ccs was driven by the T7 lac promoter, and SpyTag-β-cav1 was driven by the arabinose promoter, thus generating the plasmid pRSFDuet-ZEP-SpyCatcher-CCS-SpyTag-β-cav1. The transit peptide sequences of Cpn60α, Cpn60β and Cpn20 genes were predicted and removed, and constructed into the pACYCDuet-1 vector, and the constitutive promoter J23119 was used to replace the original T7 promoter on the vector, thereby generating the plasmid pACYCDuet-Cpn. FDHm and Fre genes were constructed into the pCDFDuet-1 vector, thereby generating the plasmid pCDFDuet-FDHm-Fre. The above genes were codon-optimized and fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. based on the host Escherichia coli. The nucleotide sequences of SpyCatcher-ccs, SpyTag-β-cav1, Cpn60α, Cpn60β, Cpn20, FDHm and Fre genes are shown in SEQ ID NO.13 to 19.

[0087] Using the synthesized gene fragment as a template, corresponding primers and P525 enzyme were used for PCR amplification to obtain a large number of target gene fragments.

[0088] Using plasmids pRSFDuet-Zep, pACYCDuet-1 and pACYCDuet-1 as templates, primers and P525 enzyme were added for PCR amplification to obtain the plasmid backbone, which was recovered using a PCR recovery kit. The target gene fragment and the plasmid backbone were connected using homologous recombination seamless splicing technology to obtain pRSFDuet-Zep-SC-CCS-ST-Cav, pACYCDuet-Cpn and pACYCDuet-FDHm-Fre plasmids.

[0089] 2. Construction of engineered bacteria producing capsanthin (1) pACYCDuet-Cpn was transferred into the chassis cell EcZea in Example (1) by the CaCl2 chemical transformation method. Subsequently, EcZeapRSFDuet-SpyTag-ZEP-SpyCatcher-CCS and pRSFDuet-ZEP-SpyCatcher-CCS-SpyTag-β-cav1 were transferred into the cell to obtain strains EcCap-1 and EcCap-2. The capsanthin production of the above two strains was compared to select a high-yielding strain. pACYCDuet-Cpn and pACYCDuet-FDHm-Fre plasmids were transferred into the high-yielding strain in sequence to obtain a recombinant engineered bacterium producing capsanthin, which was named Cap-OUC-CHB.

[0090] (2) The engineered strain Cap-OUC-CHB in (1) was used to prepare seed liquid for shake flask fermentation (fermentation time 72 h), extraction (taking the cultured fermentation liquid, centrifuging and discarding the supernatant, adding acetone to the precipitate, adding 1 mL of acetone to every 10 mL of fermentation liquid, extracting, centrifuging, filtering and removing impurities from the supernatant to obtain an extract, which contains capsanthin and capsanthin) and HPLC detection.

[0091] Different from the previous fermentation, during the fermentation, arabinose with a final concentration of 20 mM was added to the fermentation broth at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h, respectively, to determine the optimal time for scaffold protein expression, aiming to balance the folding process promoted by the molecular chaperone and the homotrimer assembly process mediated by the scaffold protein.

[0092] Output Figure 6 , Figure 7 shown. Figure 6The results showed that when ZEP and CCS were assembled, strain EcCap-1 only produced 1.23 mg / g DCW of capsanthin but no capsanthin. When CCS was artificially assembled as a homotrimer, strain EcCap-2 could produce 3.17 mg / g DCW of capsanthin and 1.01 mg / g DCW of capsanthin. In addition, under the activation of the constitutive promoter J23119, the chloroplast-derived molecular chaperone provided sufficient folding for CCS in the recombinant engineered bacteria Cap-OUC-CHB, but this was a repeated annealing process. Therefore, an optimal time was chosen during the fermentation process to add arabinose to induce the scaffold protein for the assembly of artificial homotrimers. The results showed that induction at the 36th h could not only give CCS sufficient folding time, but also allow highly active CCS to be assembled. At this time, the recombinant engineered bacteria Cap-OUC-CHB accumulated 5.15 mg / g DCW of capsanthin and 1.62 mg / g DCW of capsanthin, such as Figure 7 shown.

[0093] Based on the above determination of adding arabinose at a final concentration of 20 mM at the 36th hour, sodium formate was added to the initial culture medium (to provide a substrate for FDHm) to investigate the effect of different sodium formate concentrations on the production of capsanthin: the sodium formate concentration gradient was set to 10 mM, 15 mM, 20 mM, 25 mM and 30 mM, and the sodium formate solution was sterilized by filtering through a 0.22 μm water filter membrane and then added to the sterilized ZYP-5052 culture medium. The results are shown in Figure 8 As shown, it can be seen that when 20 mM sodium formate is added, sufficient reduced cofactors can be provided for CCS, and the accumulation of capsanthin and capsanthin is increased to 6.77 mg / gDCW and 2.18 mg / g DCW.

[0094] The present invention constructs a recombinant engineered bacterium, transforms and screens the intermediate strains for multiple times, and finally obtains a recombinant engineered bacterium Cap-OUC-CHB with high production of capsanthin. The present invention preserves the engineered bacterium, which is stored in the China Center for Type Culture Collection, and is classified and named Escherichia coli Cap-OUC-CHB, with a preservation number of CCTCC NO: M20241969, a preservation date of September 13, 2024, and a preservation address of: China Center for Type Culture Collection of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, zip code 430072.

[0095] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A capsanthin-producing Escherichia coli, characterized in that: The classification name is Escherichia coli Cap-OUC-CHB, deposited in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20241969, and the preservation date is September 13, 2024.

2. Use of the capsanthin-producing Escherichia coli according to claim 1 in the preparation of capsanthin.

3. The use according to claim 2, characterized in that: The capsanthin is selected from any one or two of capsanthin and capsorubin.

4. The use according to claim 2, characterized in that: In specific application, Escherichia coli producing capsanthin is cultured and capsanthin is extracted.

5. The use according to claim 4, characterized in that: The specific method of culturing the Escherichia coli producing capsanthin is: inoculating the seed liquid of the Escherichia coli producing capsanthin into the self-induction fermentation medium, and inducing the fermentation in a shaking table at 37° C. and 220 rpm for 48 to 96 hours.

6. The use according to claim 5, characterized in that: The auto-induction fermentation medium is selected from ZYP-5052 medium; Or: The inoculation amount of seed solution is 2%; Or: the self-induction fermentation medium contains 20 mM sodium formate; at the 36th hour of fermentation, 20 mM arabinose is added to the fermentation broth for induction.

7. The use according to claim 4, characterized in that: The specific method of extracting capsanthin is: taking the cultured fermentation liquid, centrifuging and discarding the supernatant, adding acetone to the precipitate, extracting, centrifuging, filtering and removing impurities from the supernatant to obtain an extract containing capsanthin and capsanthin.

8. The use according to claim 7, characterized in that: The amount of acetone added is: 1 mL of acetone is added to every 10 mL of fermentation liquid.

9. A method for constructing a recombinant engineering bacterium producing capsanthin, characterized in that: Here are the steps: (1) Constructing a recombinant Escherichia coli that produces zeaxanthin as a chassis cell: Using E. coli BL21 (DE3) as a starting strain, using gene editing vectors pHCY-25A and pHCY-26D to knock out the trxb gene, and then using pTrc99a as an expression vector to transfer the CrtE, CrtB, CrtI, CrtY and CrtZ genes, constructing and screening to obtain a recombinant engineered bacterium; the nucleotide sequence of N20 targeting trxB used to knock out the trxb gene is shown in SEQ ID NO.1; the nucleotide sequences of the CrtE, CrtB, CrtI, CrtY and CrtZ genes are shown in SEQ ID NOs.2 to 6, respectively; (2) Constructing a recombinant Escherichia coli that produces violaxanthin as a chassis cell: using pRSFDuet-1 as an expression vector, the Zep gene was transferred into the recombinant engineered bacteria constructed in the above step (1); then, using pTrc99a-EBIYZ as an expression vector, the Trx-m1, Trx-m2 and Trx-m4 genes were transferred to construct and screen the recombinant engineered bacteria; the nucleotide sequences of the Zep, Trx-m1, Trx-m2 and Trx-m4 genes are shown in SEQ ID NOs. 8, 10, 11 and 12, respectively; (3) Construction of recombinant Escherichia coli producing capsanthin and capsanthin: Using pRSFDuet-ZEP as an expression vector, the ccs gene with the scaffold protein SpyCatcher connected to the 5' end and the β-cav1 gene with SpyTag connected to the 5' end were transferred into the recombinant engineered bacteria constructed in the above step (2); then, using pACYCDuet-1 as an expression vector, the Cpn60α, Cpn60β and Cpn20 genes were further transferred; then, using pCDFDuet-1 as an expression vector, the FDHm and Fre genes were further transferred to construct and screen recombinant Escherichia coli producing capsanthin and capsanthin; the nucleotide sequences of the SpyCatcher-ccs, SpyTag-β-cav1, Cpn60α, Cpn60β, Cpn20, FDHm and Fre genes are shown in SEQ ID NOs. 13 to 19, respectively.

10. The recombinant engineered bacteria constructed by the construction method according to claim 9.

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